The contemporary aminoacyl-tRNA synthetases (aaRSs) are highly specific toward cognitive amino acids and tRNAs, because the fidelity of translation from genetic code to proteins relies mainly upon the accurate aminoacylation of tRNAs. Consequently, the mechanism of their recognition events is extremely complex, which has made it difficult to engineer the aaRSs with desired specificities. The central goal of this proposal is to devise a novel and practical catalytic system composed of catalytic RNA molecules (ribozymes) for the synthesis of natural and non-natural aminoacyl-tRNAs. We have recently succeeded in generating a novel ribozyme capable of synthesizing aminoacyl-tRNA. This ribozyme, referred to as ARS ribozyme (aminoacyl-tRNA synthetase ribozyme), is able to specifically recognize a glutamine cyanomethyl ester and charge the glutaminyl group on a tRNA molecule. In this proposal, we will plan to systematically evolve the ARS ribozyme into highly selective and efficient ARS ribozymes for the synthesis of various aminoacyl-tRNAs. Our ribozyme-based catalytic system will potentially offer a simple and facile method for the synthesis of aminoacyl-tRNAs in vitro. The results from these experiments will be used to drive future direction of our long-term goal, a cell-free in vitro translation system composed of ARS ribozymes, which facilitates the site-specific incorporation of non-natural amino acids into proteins. Our experiments may also provide new evidence to support an RNA-based translation system postulated in the context of the RNA world hypothesis.